scholarly journals The genetic basis of aneuploidy tolerance in wild yeast

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
James Hose ◽  
Leah E Escalante ◽  
Katie J Clowers ◽  
H Auguste Dutcher ◽  
DeElegant Robinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Treatment ◽  
Genetic Basis ◽  
Rna Binding ◽  
Laboratory Strain ◽  
Pathogenic Fungi ◽  
Wild Type ◽  
Wild Yeast ◽  
Mechanistic Understanding

Aneuploidy is highly detrimental during development yet common in cancers and pathogenic fungi – what gives rise to differences in aneuploidy tolerance remains unclear. We previously showed that wild isolates of Saccharomyces cerevisiae tolerate chromosome amplification while laboratory strains used as a model for aneuploid syndromes do not. Here, we mapped the genetic basis to Ssd1, an RNA-binding translational regulator that is functional in wild aneuploids but defective in laboratory strain W303. Loss of SSD1 recapitulates myriad aneuploidy signatures previously taken as eukaryotic responses. We show that aneuploidy tolerance is enabled via a role for Ssd1 in mitochondrial physiology, including binding and regulating nuclear-encoded mitochondrial mRNAs, coupled with a role in mitigating proteostasis stress. Recapitulating ssd1Δ defects with combinatorial drug treatment selectively blocked proliferation of wild-type aneuploids compared to euploids. Our work adds to elegant studies in the sensitized laboratory strain to present a mechanistic understanding of eukaryotic aneuploidy tolerance.

scholarly journals RNA-guided gene drives can efficiently and reversibly bias inheritance in wild yeast

2015 ◽  
Author(s):  
James E DiCarlo ◽  
Alejandro Chavez ◽  
Sven L Dietz ◽  
Kevin M Esvelt ◽  
George M Church
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Drive ◽  
Wild Type ◽  
Wild Populations ◽  
Yeast Saccharomyces Cerevisiae ◽  
Wild Yeast ◽  
Successive Generations ◽  
Gene Drives ◽  
General Method ◽  
Made In

Inheritance-biasing “gene drives” may be capable of spreading genomic alterations made in laboratory organisms through wild populations. We previously considered the potential for RNA-guided gene drives based on the versatile CRISPR/Cas9 genome editing system to serve as a general method of altering populations. Here we report molecularly contained gene drive constructs in the yeast Saccharomyces cerevisiae that are typically copied at rates above 99% when mated to wild yeast. We successfully targeted both non-essential and essential genes, showed that the inheritance of an unrelated “cargo” gene could be biased by an adjacent drive, and constructed a drive capable of overwriting and reversing changes made by a previous drive. Our results demonstrate that RNA-guided gene drives are capable of efficiently biasing inheritance when mated to wild-type organisms over successive generations.

scholarly journals Investigating the Quantitative Trait Loci Contributing to Individual Variation in Drug Response

2021 ◽  
Author(s):  
◽  
Christina Roberts
Keyword(s):  
Quantitative Trait Loci ◽  
Drug Treatment ◽  
Individual Variation ◽  
Quantitative Trait ◽  
Drug Response ◽  
Genetic Basis ◽  
Laboratory Strain ◽  
High Dose ◽  
Trait Loci ◽  
Insight Into

<p>Individuals often display a wide variety of phenotypic responses to drug treatment, in terms of both efficacy and side effects. Part of this variation appears to have an individual genetic basis which is not well understood. It is well established in the literature that most traits, including drug response, are not controlled by a single gene, but rather arise from multiple loci known as quantitative trait loci (QTL). This thesis investigated the genetic basis of individual variability of response to two antifungal agents whose targets are known—namely benomyl (an industrial fungicide) and ketoconazole (a medicinal fungicide). A collection of 33 Saccharomyces cerevisiae yeast strains, sourced from the Saccharomyces Genome Resequencing Project (SGRP, Sanger Institute) was used to model individuals as these strains carry natural variation in terms of single nucleotide polymorphisms (SNPs) akin to human individuals.  Drug response measurements using serial spot dilution and high-throughput 384-colony robotic pinning screens were used to select four SGRP strains on the basis of drug resistance or sensitivity relative to the laboratory strain BY. These were L-1374 that was sensitive to benomyl compared to BY; UWOPS87-2421 that was resistant to benomyl compared to BY; Y12 that was sensitive to ketoconazole compared to BY; DBVPG6044 that was resistant ketoconazole compared to BY. The four strains described were crossed individually with the BY laboratory strain and the resultant diploids were sporulated to obtain meiotic recombinant offspring. Spores were then subjected ten cycles of intercrossing in order to obtain advanced intercross lines (AILs); these contain reduced linkage disequilibrium between marker and trait genomic position and act to refine the localising potential of the QTL. The segregant offspring produced following the setup of AIL were subjected to studies to investigate the heritability of drug response to intermediate and high dose of benomyl or ketoconazole. It was concluded that in each of the crosses trialled, the drug response was a multigenic trait. Furthermore, the broad sense heritability estimates were high (L-1374×BY: H² = 0.91 and 0.92 for response to 75 μM and 137.5 μM benomyl respectively; UWOPS87-2421×BY: H² = 0.75 and 0.87 for response to 150 μM and 250 μM benomyl; Y12×BY: H² = 0.9 and 0.88 for response to 60 μM and 100 μM ketoconazole). This indicates that most of the variance seen in drug response arises due to genetic variance. Additionally, the relative drug sensitivity in each of the crosses trialled was found to be either a dominant trait (either partially or fully so).  Finally QTL mapping through next generation sequencing bulk segregant analysis (NGS-BSA) confirmed the multigenic nature of the drug response in the selected strains. The effect of intermediate versus high dose drug treatment revealed that the QTL network is largely conserved between treatment regimens (L-1374×BY cross: three and five QTL upon treatment with 30 μM and 50 μM benomyl respectively; UWOPS87-2421×BY cross: nine and 18 QTL upon treatment with 45 μM and 80 μM of benomyl; Y12×BY cross: 41 and 56 QTL for response to 11.5 μM and 15 μM of ketoconazole; DBVPG6044×BY cross: 12 and 10 QTL for the response to 25 μM and 65 μM ketoconazole). In order to investigate the contribution of individual variation to drug response, the QTL network of the sensitive and the resistant strain for each drug were compared. It was revealed that although there is a conserved core of QTL for response to benomyl and ketoconazole respectively, the individual strains possess a considerable number of strain-specific QTL. This suggested that individual variation may indeed play a significant role in drug response. Analysis of the top-ranking QTL (in terms of LOD score) for each of the four strains revealed that each of them harboured genes that have literature-supported relationships to their relevant drug.  This thesis presents a significant contribution to existing literature in terms of elucidating the QTL network underlying individual response to benomyl and ketoconazole. The findings from this study have practical potential to provide improved insight into factors that can produce antifungal resistance (a growing and significant clinical problem). Furthermore, it provides insight into better therapeutic regimens that can improve medicinal treatment for individuals.</p>

scholarly journals Investigating the Quantitative Trait Loci Contributing to Individual Variation in Drug Response

2021 ◽  
Author(s):  
◽  
Christina Roberts
Keyword(s):  
Quantitative Trait Loci ◽  
Drug Treatment ◽  
Individual Variation ◽  
Quantitative Trait ◽  
Drug Response ◽  
Genetic Basis ◽  
Laboratory Strain ◽  
High Dose ◽  
Trait Loci ◽  
Insight Into

<p>Individuals often display a wide variety of phenotypic responses to drug treatment, in terms of both efficacy and side effects. Part of this variation appears to have an individual genetic basis which is not well understood. It is well established in the literature that most traits, including drug response, are not controlled by a single gene, but rather arise from multiple loci known as quantitative trait loci (QTL). This thesis investigated the genetic basis of individual variability of response to two antifungal agents whose targets are known—namely benomyl (an industrial fungicide) and ketoconazole (a medicinal fungicide). A collection of 33 Saccharomyces cerevisiae yeast strains, sourced from the Saccharomyces Genome Resequencing Project (SGRP, Sanger Institute) was used to model individuals as these strains carry natural variation in terms of single nucleotide polymorphisms (SNPs) akin to human individuals.  Drug response measurements using serial spot dilution and high-throughput 384-colony robotic pinning screens were used to select four SGRP strains on the basis of drug resistance or sensitivity relative to the laboratory strain BY. These were L-1374 that was sensitive to benomyl compared to BY; UWOPS87-2421 that was resistant to benomyl compared to BY; Y12 that was sensitive to ketoconazole compared to BY; DBVPG6044 that was resistant ketoconazole compared to BY. The four strains described were crossed individually with the BY laboratory strain and the resultant diploids were sporulated to obtain meiotic recombinant offspring. Spores were then subjected ten cycles of intercrossing in order to obtain advanced intercross lines (AILs); these contain reduced linkage disequilibrium between marker and trait genomic position and act to refine the localising potential of the QTL. The segregant offspring produced following the setup of AIL were subjected to studies to investigate the heritability of drug response to intermediate and high dose of benomyl or ketoconazole. It was concluded that in each of the crosses trialled, the drug response was a multigenic trait. Furthermore, the broad sense heritability estimates were high (L-1374×BY: H² = 0.91 and 0.92 for response to 75 μM and 137.5 μM benomyl respectively; UWOPS87-2421×BY: H² = 0.75 and 0.87 for response to 150 μM and 250 μM benomyl; Y12×BY: H² = 0.9 and 0.88 for response to 60 μM and 100 μM ketoconazole). This indicates that most of the variance seen in drug response arises due to genetic variance. Additionally, the relative drug sensitivity in each of the crosses trialled was found to be either a dominant trait (either partially or fully so).  Finally QTL mapping through next generation sequencing bulk segregant analysis (NGS-BSA) confirmed the multigenic nature of the drug response in the selected strains. The effect of intermediate versus high dose drug treatment revealed that the QTL network is largely conserved between treatment regimens (L-1374×BY cross: three and five QTL upon treatment with 30 μM and 50 μM benomyl respectively; UWOPS87-2421×BY cross: nine and 18 QTL upon treatment with 45 μM and 80 μM of benomyl; Y12×BY cross: 41 and 56 QTL for response to 11.5 μM and 15 μM of ketoconazole; DBVPG6044×BY cross: 12 and 10 QTL for the response to 25 μM and 65 μM ketoconazole). In order to investigate the contribution of individual variation to drug response, the QTL network of the sensitive and the resistant strain for each drug were compared. It was revealed that although there is a conserved core of QTL for response to benomyl and ketoconazole respectively, the individual strains possess a considerable number of strain-specific QTL. This suggested that individual variation may indeed play a significant role in drug response. Analysis of the top-ranking QTL (in terms of LOD score) for each of the four strains revealed that each of them harboured genes that have literature-supported relationships to their relevant drug.  This thesis presents a significant contribution to existing literature in terms of elucidating the QTL network underlying individual response to benomyl and ketoconazole. The findings from this study have practical potential to provide improved insight into factors that can produce antifungal resistance (a growing and significant clinical problem). Furthermore, it provides insight into better therapeutic regimens that can improve medicinal treatment for individuals.</p>

scholarly journals Overexpression of Sbe2p, a Golgi Protein, Results in Resistance to Caspofungin in Saccharomyces cerevisiae

2002 ◽  
Vol 46 (8) ◽  
pp. 2462-2469 ◽  
Author(s):  
Nir Osherov ◽  
Gregory S. May ◽  
Nathaniel D. Albert ◽  
D. P. Kontoyiannis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Restriction Analysis ◽  
Pathogenic Fungi ◽  
Cdna Clones ◽  
Wild Type ◽  
Transport Pathway ◽  
Synthetic Complete ◽  
Direct Demonstration ◽  
Golgi Protein

ABSTRACT Caspofungin inhibits the synthesis of 1, 3-β-d-glucan, an essential cell wall target in fungi. Genetic studies in the model yeast Saccharomyces cerevisiae have shown that mutations in FKS1 and FKS2 genes result in caspofungin resistance. However, direct demonstration of the role of gene overexpression in caspofungin resistance has been lacking. We transformed wild-type S. cerevisiae with an S. cerevisiae URA3-based GAL1 cDNA library and selected transformants in glucose synthetic complete plates lacking uracil (glucose SC minus uracil plates). We then moved the transformants to galactose SC minus uracil plates containing caspofungin (1 μg/ml) and looked for caspofungin-resistant colonies. We retested the candidates (true positives were sensitive on glucose caspofungin and resistant on galactose caspofungin media, respectively). We identified 16 caspofungin-resistant candidates. Restriction analysis and hybridization confirmed that 15 of the 16 clones were identical. We sequenced one of the cDNA clones and found that it contained the cDNA for SBE2. SBE2 has been described in S. cerevisiae to encode a Golgi protein involved in the transport of cell wall components (B. Santos and M. Snyder, Mol. Biol. Cell, 11:435-452, 2000). The SBE2 cDNA plasmid conferred again galactose-dependent caspofungin resistance when transformed back into the wild-type S. cerevisiae. Finally, the SBE2 deletion mutant was hypersensitive to caspofungin. In conclusion, overexpression of Sbe2p under the regulated control of the GAL1 promoter results in caspofungin resistance in S. cerevisiae. This transport pathway may provide insight into the tolerance or lack of sensitivity to caspofungin of some pathogenic fungi.

scholarly journals ABC Transporters and Azole Susceptibility in Laboratory Strains of the Wheat Pathogen Mycosphaerella graminicola

2002 ◽  
Vol 46 (12) ◽  
pp. 3900-3906 ◽  
Author(s):  
Lute-Harm Zwiers ◽  
Ioannis Stergiopoulos ◽  
Johannes G. M. Van Nistelrooy ◽  
Maarten A. De Waard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Rhodamine 6G ◽  
Laboratory Strain ◽  
Mycosphaerella Graminicola ◽  
Cross Resistance ◽  
Wild Type ◽  
Wild Type Level ◽  
Abc Transporter Genes

ABSTRACT Laboratory strains of Mycosphaerella graminicola with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both. Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole. No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes MgAtr1 to MgAtr5 and the sterol P450 14α-demethylase gene, CYP51. ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of Saccharomyces cerevisiae and disruption of MgAtr1 in one specific M. graminicola laboratory strain with constitutive MgAtr1 overexpression restored the level of susceptibility to cyproconazole to wild-type levels. However, the level of accumulation in the mutant with an MgAtr1 disruption did not revert to the wild-type level. We propose that variations in azole susceptibility in laboratory strains of M. graminicola are mediated by multiple mechanisms.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

scholarly journals INTRACISTRONIC MAPPING OF ELECTROPHORETIC SITES IN DROSOPHILA MELANOGASTER: FIDELITY OF INFORMATION TRANSFER BY GENE CONVERSION

Genetics ◽  
1974 ◽  
Vol 76 (2) ◽  
pp. 289-299
Author(s):  
Margaret McCarron ◽  
William Gelbart ◽  
Arthur Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Information Transfer ◽  
Large Scale ◽  
Genetic Basis ◽  
Xanthine Dehydrogenase ◽  
Specific Protein ◽  
Wild Type ◽  
Electrophoretic Variation ◽  
The Stability ◽  
Recombination Experiments

ABSTRACT A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster. A number of wild-type isoalleles of the rosy locus have been isolated which are associated with the production of electrophoretically distinguishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a nonselective marker in the cross. Additionally, a large-scale recombination experiment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of crossover and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic character, this observation argues against a special mutagenesis hypothesis to explain conversions resulting from allele recombination studies.

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